This invention relates to methods for the use of morphogenically active fragments of 60A protein to induce tissue morphogenesis in mammals, including methods for promoting tissue stasis, repair and regeneration, and methods for increasing progenitor cell populations using morphogenically active fragments of the protein.
Cell differentiation is the central characteristic of morphogenesis which initiates in the embryo, and continues to various degrees throughout the life of an organism in adult tissue repair and regeneration mechanisms. The degree of morphogenesis in adult tissue varies among different tissues and is related, among other things, to the degree of cell turnover in a given tissue. On this basis, tissues can be divided into three broad categories: (1) tissues with static cell populations such as nerve and skeletal muscle where there is no cell division and most of the cells formed during early development persist throughout adult life; (2) tissues containing conditionally renewing populations such as liver where there is generally little cell division but, in response to an appropriate stimulus, cells can divide to produce daughters of the same differentially defined type; and (3) tissues with permanently renewing populations including blood, testes and stratified squamous epithelia which are characterized by rapid and continuous cell turnover in the adult. Here, the terminally differentiated cells have a relatively short life span and are replaced through proliferation of a distinct subpopulation of cells, known as stem or progenitor cells.
The cellular and molecular events which govern the stimulus for differentiation of these cells is an area of intensive research. In the medical field, it is anticipated that the discovery of factor(s) which control cell differentiation and tissue morphogenesis will advance significantly medicine's ability to repair and regenerate diseased or damaged mammalian tissues and organs. Particularly useful areas include reconstructive surgery and in the treatment of tissue degenerative diseases including arthritis, emphysema, osteoporosis, cardiomyopathy, cirrhosis, and degenerative nerve diseases.
A number of different factors have been isolated in recent years which appear to play a role in cell differentiation.
Recently, various members of the structurally related proteins of the transforming growth factor (TGF)-.beta. superfamily of proteins have been identified as true morphogens.
This "family" of proteins, sharing substantial amino acid sequence homology within their morphogenically active C-terminal domains, including a conserved six or seven cysteine skeleton, are capable of inducing tissue-specific morphogenesis in a variety of organs and tissues, including bone, cartilage, liver, dentin, periodontal ligament, cementum, nerve tissue and the epithelial mucosa of the gastrointestinal tract (see the copending, related U.S. applications Ser. No. 667,274, filed Mar. 11, 1991 abandoned in favor of (U.S. application Ser. No. 08/404,113 filed, Mar. 14, 1995) and U.S. application Ser. No. 08/404,113 filed Mar. 14, 1995 and U.S. application Ser. No. 08/260,675, filed Jun. 16, 1994 and U.S. application Ser. No. 08/132,883 filed May 21, 1995. The proteins apparently bind to surface receptors or otherwise contact and interact with progenitor cells, predisposing or stimulating the cells to proliferate and differentiate in a morphogenically permissive environment. The morphogens are capable of inducing the developmental cascade of cellular and molecular events that culminate in the formation of new organ-specific tissue, including any vascularization, connective tissue formation, and nerve ennervation as required by the naturally occurring tissue.
Among the proteins useful in tissue morphogenesis are proteins originally identified as bone inductive proteins, such as the OP-1 (comprising, e.g. Seq. ID NO. 3), OP-2 (comprising, e.g. Seq. ID NO. 5) and the CBMP2 (comprising, e.g. Seq. ID NO. 7 and 8) proteins, as well as amino acid sequence-related proteins such as BMP5 (comprising, e.g., Seq. ID NO. 14) and BMP6 (comprising, e.g., Seq. ID NO. 15), DPP (comprising, e.g., Seq. ID NO. 9) (from Drosophila), Vgl (comprising, e.g., Seq. ID NO. 10) (from Xenopus), and Vgr-1 (comprising, e.g., Seq. ID NO. 11) and GDF-1 (comprising, e.g., Seq. ID NO. 12) (from mouse see, for example, application Ser. No. 08/404,113, filed Mar. 14, 1995, U.S. Ser. No. 667,274 (abandoned in favor of CIP U.S. application Ser. No. 08/404,113, filed Mar. 14, 1995 and application Ser. No. 08/432,883 filed May 2, 1995. These TGF-.beta. superfamily members comprise a distinct subfamily of proteins different from other members of the TGF-.beta. superfamily in that the family of morphogenic proteins are able to induce the full cascade of events that result in tissue morphogenesis, including stimulating cell proliferation and cell differentiation, supporting the growth and maintenance of differentiated cells and inducing the "redifferentiation" of transformed cells to display a morphology characteristic of untransformed cells. The morphogenic proteins apparently act as endocrine factors rather than as local-acting growth factors like TGF-.beta.. Specifically, the endogenous morphogens may be synthesized and secreted from a factor-producing tissue and can be transported to, and act on, a tissue at a distance, e.g., a tissue other than the tissue in which they are synthesized.
The morphogens are synthesized in the cell as a precursor molecule approximately three times larger than the mature protein that is processed to yield mature disulfide-linked dimers comprising the C-terminal domain of the precursor sequence. The proteins are inactive when reduced, but are active as oxidized homodimeric species as well as when oxidized in combination with other morphogens to produce heterodimers. The proteins useful in tissue morphogenesis typically require a suitable environment enabling cells to proliferate and differentiate in a tissue-specific manner into, e.g., bone-producing osteoblasts, hemopoietic cells, or liver cells, depending on the nature of the local environment. The proliferation and differentiation of cells induced by the morphogenic proteins requires a suitable local environment including a suitable substratum on which the cells can anchor. The proliferating and differentiating cells also require the presence of appropriate signals to direct their tissue-specificity, such as cell surface markers.
Recently, another member of the TFG-.beta. superfamily of structurally related proteins has been identified in Drosophila melanogaster, the Drosophila 60A gene (comprising, e.g., Seq. ID NO. 1) (Wharton et al., (1991) Proc. Nat'l. Acad. Sci. USA 88: 9214-9218.) Northern blot analysis of Drosophila tissue with a C-terminal 60A-specific probe suggests that the 60A gene (comprising, e.g., Seq. ID NO. 1) is expressed throughout development with peaks of transcription during early embryogenesis. The 60A gene (comprising, e.g., Seq. ID NO. 1) consists of a single exon comprising a 1365 base-pair open reading frame encoding a 455 amino acid protein. It has been discovered that the encoded amino acid sequence for 60A (comprising, e.g., Seq. ID NO. 2) includes regions sharing high sequence homology with members of the family of morphogenic proteins.
It is an object of this invention to provide methods for utilizing a morphogenically active fragment of the 60A protein (comprising, e.g., Seq. ID NO. 2) to induce the developmental cascade of tissue morphogenesis for a variety of tissues in mammals. The morphogenic properties of 60A protein (comprising, e.g., Seq. ID NO. 2) include the ability to induce proliferation and differentiation of progenitor cells, and the ability to support and maintain the differentiated phenotype through the progression of events that results in the formation of adult tissue. Another object is to provide methods for the expression and isolation of a morphogenically active fragment of 60A protein (comprising, e.g., Seq. ID NO. 2) using recombinant DNA techniques. Still another object is to provide tissue-specific acellular matrices that may be used in combination with a morphogenically active fragment of 60A protein (comprising, e.g., Seq. ID NO. 2), and methods for their production. Other objects include utilizing a morphogenically active fragment of 60A protein (comprising, e.g., Seq. ID NO. 2) in a range of methods, including methods for increasing a progenitor cell population in a mammal; methods for stimulating progenitor cells to differentiate in vivo or in vitro and to maintain their differentiated phenotype; methods for inducing tissue-specific growth in vivo, and methods for the replacement of diseased or damaged tissue in vivo. These and other objects and features of the invention will be apparent from the description, drawings, and claims which follow.